The epithelial Na+ channel (ENaC) forms a pathway for Na+ absorption in the kidney, lung, and other epithelia. ENaC is tightly regulated in order to maintain Na+ homeostasis and to control blood pressure. Defects in this regulation upset the delicate balance and cause disease. Importantly, many of the identified inherited forms of hypertension result from defects in ENaC regulation. A defect in ENaC regulation may also contribute to the pathogenesis of cystic fibrosis. Thus, our long term goal is to understand the mechanisms that regulate ENaC as a prerequisite for the development of more targeted treatments for these diseases. A convergence of recent discoveries has focused attention on ubiquitination as a critical mechanism that regulates ENaC trafficking. By reducing ENaC surface expression, ubiquitination decreases epithelial Na+ absorption, which is an important adaptive response in the face of Na+/volume excess. In this proposal, we will investigate the molecular mechanisms by which ubiquitination controls ENaC trafficking. This work capitalizes on our previous discoveries that the E3 ubiquitin ligase Nedd4-2 catalyzes ENaC ubiquitination and reduces ENaC expression at the cell surface at two distinct steps in the trafficking pathway (endocytosis and lysosomal degradation). Importantly, defects in this pathway are responsible for Liddle's syndrome. Moreover, Nedd4-2 is a critical convergence point by which aldosterone and vasopressin regulate ENaC. Although it is clear that ubiquitination plays a critically important role in the regulation of epithelial Na+ absorption, there are important gaps in our knowledge. The overall goal of this proposal is to understand the molecular mechanisms by which ubiquitination regulates ENaC, and hence, epithelial Na+ transport. We will investigate mechanisms by which E3 ubiquitin ligases regulate ENaC trafficking, investigate mechanisms that regulate ENaC sorting in endosomes, and investigate the role of lysine acetylation in ENaC trafficking. By testing novel hypotheses and using innovative approaches, this work will generate new insights into mechanisms by which ubiquitination regulates ENaC surface expression, and hence, epithelial Na+ transport and Na+ homeostasis. This may have important implications for our understanding and treatment of diseases including hypertension and cystic fibrosis.